CRISPR/Cas9 technology in the modeling of and treatment of mucopolysaccharidosis

Mucopolysaccharidosis (MPS) syndromes are a group of heterogeneous genetic disorders in terms of genetic basis and clinical manifestations, ranging from mild to fatal forms. There are a number of applied or prospective treatment modalities for MPS, including bone marrow transplantation, enzyme replacement therapy, targeted gene therapy and substrate reduction therapy. Recently, CRISPR/Cas9 technology has emerged as a novel tool for several metabolic disorders, such as MPS. This review concentrates on the application of this technique in the treatment of MPS, particularly MPS I, and modeling of disease-causing mutations.


Introduction
Mucopolysaccharidosis (MPS) syndromes are a group of heterogeneous genetic disorders comprising 7 types and 13 subgroups [1].All of these conditions are caused by defects in the enzymes that degrade glycosaminoglycans (GAGs).These defects result in the widespread accumulation of GAGs within the lysosomes of different organs.Therefore, clinical manifestations can be detected in almost all body systems, such as eye, central nervous system, lung, heart, bone and the gastrointestinal system [2].While some cases are presented with minor systemic and ocular defects and have a normal life span, other might have severe phenotypes resulting in the death in the first few months of life [1].
All types of MPS except for type II are inherited as autosomal recessive disorders [3].Alternatively named as the Hunter's syndrome, MPS type II has an X-linked inheritance and is caused by the defects in the iduronate-2-sulfatase enzyme encoded by IDS gene on chromosome Xq28 [3].Type I MPS is caused by mutations in IDUA gene, which is located on chromosome 4p16, and encodes the a-L-iduronidase enzyme.This type of MPS has three subtypes, namely Hurler, Hurler-Scheie, and Scheie syndromes, with the first subtype being the most severe subtype [4].Accumulation of dermatan and heparan sulfates in MPS I occurs in various tissues and results in pervasive organ dysfunction [4].
There are a number of treatment modalities, including bone marrow transplantation and enzyme replacement therapy to increase their life span of affected individuals and enhance their quality of life [1].However, application of enzyme replacement therapy is limited by the challenges caused by crossing the blood-brain barrier [11].Thus, this treatment is not applicable in the severe forms of MPS with the involvement of the central nervous system.Targeted gene therapy and substrate reduction therapy have also been suggested as prospective treatments for MPS [1].Recently, CRISPR/Cas9 technology has emerged as a novel tool for several metabolic disorders [12].This review focuses on the application of this technique in the treatment of MPS and modeling of disease-causing mutations.

CRISPR/Cas9 technique
Being firstly recognized as an RNA-mediated immune system, CRISPR/Cas system defends prokaryotes against bacteriophages and horizontal plasmid transmission [13].Generally, the CRISPR/Cas systems are categorized into two main subtypes.While class 1 systems use multi-protein effector complexes, class 2 systems implement single-effector complexes making them more suitable for gene editing applications and screening [14].The CRISPR/Cas9 system is an example of the second class, which has been broadly utilized for gene editing applications.Cas9 protein and guide RNA (gRNA) are the main [31] M. Reyhani-Ardabili and S. Ghafouri-Fard apparatuses of the CRISPR/Cas9 system.In fact, Cas9 is a multi-domain DNA endonuclease capable of cleaving the target DNA and making double-strand breaks [15].Mechanistically, gRNA is made by the combination of tracrRNA and crRNA.While the former serves as a binding scaffold for nuclease, the latter pairs with the target sequence and is responsible for its specificity [16].Thus, gRNA can be programmed to provide the specificity of the CRISPR/Cas9 system.The action of this system is accomplished through three steps of recognition, cleavage and restoration of induced double strand breaks.The latter is performed by the host cellular system through non-homologous end-joining or homology-directed repair (HDR) pathway with the latter being more accurate [17].Numerous in vitro and in vivo approaches have demonstrated applicability of CRISPR/Cas9 systems for modeling and treatment of different types of MPS.

CRISPR/Cas9 systems for modeling of different types of MPS
Several intracellular mechanisms contribute to the pathogenesis of different types of MPS.Although a number of related cascades have been identified, details about early cellular aberrations that lead to irreversible neuronal injury have not been elucidated.CRISPR/Cas9 technology can facilitate development of cellular models of MPS and identification of the cellular cascades leading to certain abnormalities.Badenetti et al. used this technology to develop two human neuronal cell lines with IDS loss of function.They designed sgRNA against IDS exon 4. The first clone had an 18 nucleotide deletion in the mentioned exon and the second one carried a 203 nucleotide deletion including twenty nucleotides of this exon.Neuronal cells carrying these mutations had no IDS enzymatic activity and exhibited high GAG storage which led to reduced differentiation, down-regulation of LAMP1 and RAB7 proteins, compromised lysosomal acidification and augmented lipid storage.Furthermore, one of the two clones exhibited low levels of the autophagic marker p62.However, none of them exhibited noticeable oxidative stress or mitochondrial morphological changes.Thus, impaired IDS activity was suggested to affect neuronal differentiation at cellular level [18].

CRISPR/Cas9 technology for treatment of MPS I
de Carvalho et al. used the CRISPR-Cas9 editing system for correction of the most common MPS I-related mutation.In vitro assessments revealed enhancement of IDUA activity and reduction of lysosomal mass in human fibroblasts homozygous for p.Trp402*.Moreover, the presence of wildtype sequence in these cells was confirmed by next generation sequencing, revealing the ability of CRISPR-Cas9 genome editing for correction of causative mutations in MPS I [19].This treatment strategy was also tested in vivo.In a combined in vitro and in vivo experiment, Schuh et al. used cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for MPS I gene editing.These complexes could significantly increase IDUA activity and reduce lysosomal abnormalities in fibroblasts of MPS I patients.Besides, hydrodynamic injection of the liposomal complexes in newborn MPS I mice resulted in significant enhancement of serum IDUA level.These complexes were detected in the lungs and heart.Besides, cardiovascular parameters were improved in animals after treatment with the liposomal formulation [20].
In another CRISPR/Cas9-based experiment, IDUA was introduced in the hematopoietic stem cells under the govern of a robust ubiquitous promoter.This gene was hosted in the CCR5 safe-harbor locus.Transplantation of these cells into a mouse model of MPS I led to reduction, but not normalization of neuroinflammation, and elimination of GAG in the liver and spleen.Yet, this treatment did not lead to clearance of GAG from the brain.While some behavioral abnormalities were amended, working memory was lessened in the treated animals [21].
Ou et al. made a proprietary system that inserted a promoterless IDUA cDNA sequence into the locus encoding albumin in the hepatocytes.They used adeno-associated virus-8 (AAV8) vector for delivery of this system into neonatal and adult MPS I mice.They showed enhancement of IDUA enzyme activity in the brain, normalization of storage levels, and improvement of memory and learning ability as demonstrated by neurobehavioral tests.Besides, histological test showed the efficacy of this method in reduction of foam cells in the hepatic tissue and vacuolation in neurons.Their experiments caused no vector-associated toxicity, no increased tumorigenesis and no off-target effects.The latter was confirmed through the unbiased genome-wide sequencing [22].
Ibraheim et al. described AAV structures that express Nme2Cas9 and either two sgRNAs, or a single sgRNA.While the former was used for segmental deletion, the latter was designed to be a template for HDR.[33] (continued on next page) M. Reyhani-Ardabili and S. Ghafouri-Fard They also used anti-CRISPR proteins to permit self-inactivation of vectors via Nme2Cas9 cleavage.The designed strategy was able to treat MPS I in mice through HDR-based editing method.Authors concluded that single-vector AAVs can be used to yield diverse therapeutic genome editing results [23].
Most notably, an experiment in mouse models of MPS I showed that nasal administration of liposomal complexes transporting two plasmids that encode the CRISPR/Cas9 system and the IDUA gene resulted in a moderate enhancement of IDUA activity in the lung, heart, and brain.Moreover, this treatment could reduce GAG concentrations in the serum, urine, tissues, and brain cortex.Besides, authors documented improvement in behavioral tests in the treated animals [24].Table 1 shows the results of different attempts for MPS modeling using CRISPR/Cas9.

CRISPR/Cas9 technology for treatment of other types of MPS
Leal et al. showed the suitability of a CRISPR/Cas9 method for treatment of MPS IV through using a Cas9 nickase to insert an expression cassette encompassing GALNS cDNA into the AAVS1 locus in human fibroblasts of MPS IVA patients.Notably, they used iron oxide nanoparticles as non-viral vectors [32].They experiments showed long-term expression of the desired gene, reduction of lysosomal mass, and amelioration of mitochondrial-derived reactive oxygen species in the mentioned fibroblasts [32].Subsequently, they used the similar method in mouse model of MPS IVA through inserting the human GALNS cDNA into the ROSA26 locus.They demonstrated improved GALNS activity, mono-keratan sulfate decrease, and partial amelioration of the bone pathology.Thus, they provided in vivo proof of the capacity of a CRISPR/nCas9-based method for treatment of MPS IVA using non-viral vectors [33].
As one of the pioneer studies in this field, extracellular vesicles were used to deliver CRISPR genome editing in Gusb mice as a mouse model of human MPS VII.AAV2-DJ vectors were administered to the mice through tail vein or retroorbital intravenous infusion.The latter treatment was able to improve both corneal transparency and survival of the experimental mice [34].
Fig. 1 shows an overview of different MPS_related sites edited by CRISPR/Cas9 in different species.
Table 2 summarizes the results of studies that reported the application of CRISPR/Cas9 technology for editing of genes related to different types of MPS.

Discussion
Since MPS subtypes are monogenic diseases, gene therapy-based strategies are regarded as promising treatment methods as they are supposed to deliver long-term expressions of the desired transgene.The results of first clinical trial of genome editing via AAV-zinc-finger nucleases for MPS I/II showed promising safety profile with indications of targeted genome editing in liver.Yet, no long-term enzyme expression was documented in the blood [40].Thus, this field awaits novel in vivo genome editing strategies or alternative delivery systems.
The implementation of CRISPR/Cas9 in MPS has raised the hope for the treatment of MPS, particularly for those who are not benefited from routine therapeutic options.Most of ex vivo and in vivo platforms have been examined in the contexts of MSP I, yet other types of MPS can also benefit from these techniques.In vivo application of these techniques awaits assessment of the safety of therapeutic genome editing systems [41].These systems might induce modifications at unplanned genomic sites that could eventually lead to tumorigenicity.Short-lived Cas9 and mutant Cas9 with higher fidelity have shown to be effective in reduction of abrogation of this off-target problem [21,42,43].Moreover, in silico tools have facilitated recognition of off-target sites.For instance, Carneiro et al. evaluated possible off-targets for a sgRNA targeting p. Trp402* as the most common variant detected in MPS I patients.They reported 272 potential off-target sequences as well as 84 polymorphic M. Reyhani-Ardabili and S. Ghafouri-Fard sites.Notably, most of polymorphic sites were predicted to reduce the probability of off-target cleavage.They also created a new PAM based on this analysis.Thus, off-targets should be screened in a population-specific context to increase safety and efficiency of CRISPR/Cas9-based methods [44].Another challenging issue is the selection of desirable vectors.Although viral vectors have several benefits, their application is limited by some challenging issues, including the possibility of induction of immune response, oncogenesis activation, low package capacity, and vector dilution [45].Non-viral vectors, such as iron oxide nanoparticles offer an alternative method with promising results in animal models [33].Notably, these nanoparticles demonstrated high biocompatibility because they can be metabolized by the iron metabolism pathway [46].Moreover, these nanoparticles may be proposed as a solution for the challenge encountered by the presence of neutralizing antibodies against Cas9 [33].Table 3 summarizes some drawbacks when working with the CRISPR/Cas9 system and possible solutions.
Direct gene editing systems introduce only one functional copy of the desired gene into target cells, so they lack sufficient efficacy for treatment of diseases [47].Overexpression the desired gene in hematopoietic stem cells is another strategy that might be used in future.Alternatively, application of a safe harbor locus to overexpress the desired gene has been suggested as a gene editing strategy in MPS.This approach led to promising results in the MPS I mouse models [21].
Taken together, CRISPR/Cas9 technique offers a promising method for the treatment of MPS.However, further assays using mice models of MPS are needed to test the real therapeutic effect of CRISPR/Cas9 approach in this type of metabolic disorders.Moreover, the minimum enzyme activity needed for amelioration of pathologic events in each tissue as well as the enzyme activity yield following different delivery methods should be determined.Besides, the presence of specific anti-Cas9 antibodies or cytotoxic T cells as well as the proinflammatory profile should be investigated in animal models treated with CRISPR/ Cas9-based methods.

Funding
No funding was received.

Declaration of competing interest
Authors declare no conflict of interests.

Table 2
Application of CRISPR/Cas9 technology for editing of genes related to different types of MPS.

Table 3
Drawbacks of the CRISPR/Cas9 system and possible solutions.